Abstract Mucus is the primary material that mediates interactions with the outside world in organisms from humans to jellyfish. Mucus gels function as a hydrating barrier involved in events such as embryo implantation, absorption of nutrients, drugs, and pathogens, while also housing the majority of the microbiome. Despite these essential roles, mucus composition, physical properties, and biology remain poorly defined. This is because the major component, mucin glycoproteins, is innately heterogeneous and cannot be reproducibly obtained by any current methodology. This roadblock has hindered our understanding of epithelial biology across diverse fields. The overall objective of this proposal is to generate synthetic mucus as transformative materials to probe the structure and function of native mucus, and with biomedical applications treating compromised tissues. We hypothesize that synthetic multi-block glycopolypeptides can emulate natural multi-domain gel-forming mucins, but with precisely defined and tunable compositions capable of selective modulation of gel properties and bioactivity. Native mucins are a family of 20+ glycoproteins characterized by massive rod-like domain rich in glycosylated-Ser/Thr, and short terminal domains that play a role in formation of cross-linked mucins bundles via Cys disulfides and hydrophobic interactions. Mucin expression and splice variation are unique to each tissue and disease, and the proteins' glycosylation patterns are the product of complex metabolic pathways controlled by >1000 genes. These pathways are poorly understood and cannot be manipulated by any current genetic or biochemical methods. Overall, biological mucins are too heterogeneous to probe many specific hypotheses. Glycopolymers have been explored as mucus-mimics, but prior examples have failed to recapitulate the chemical structures and biophysics of native mucins. During the project period, we will 1) develop tunable and reproducible synthetic mucins based on multi- block glycopolypeptides that faithfully emulate the chemical and biophysical properties of natural mucins, and 2) unravel how mucus composition (pH, ions, lipids, DNA, proteins) affects both gel physical properties and glycan-dependent bioactivity. We will precisely tune the glycan patterns by chemical synthesis and enzymatic glycosylation to prepare binding or control ligands to interact with glycan-binding proteins. These properties cannot be controlled by any other current methods. We will assemble the glycopolypeptides into gels with varied compositions inspired by analysis of native mucus, and we will benchmark our materials against commercially available mucins. We expect to provide new tools for our lab and others to study previously untestable hypotheses regarding mucosal transport and biology relevant to health and disease. Success of the proposed research is anticipated to make a transformative impact across diverse fields from materials science and glycobiology to pharmaceutics, immunology, infectious diseases, gasteroenterology, and gynecology.